Materials and methods for profiling micrornas

ABSTRACT

The present invention provides materials and methods for detecting, quantifying, and/or high-throughput-profiling microRNAs. Advantageously, the present invention is more sensitive and specific than other currently-available miRNA qPCR assays. In addition, the present invention is convenient, easy-to-perform, and cost-effective. In one embodiment, the present invention provides a universal primer for reverse transcription of all miRNAs, a universal reverse primer for PCR amplification reaction, and universal probes. In another embodiment, the present invention provides assays that allow simultaneous detection and/or quantification of a plurality of target miRNAs using a single reverse transcription reaction.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application a divisional application of U.S. applicationSer. No. 14/000,517, filed Aug. 20, 2013, which is the National Stage ofInternational Application No. PCT/US2012/029522, filed Mar. 16, 2012,which claims the benefit of U.S. Provisional Application Ser. No.61/454,334, filed Mar. 18, 2011, each of which is hereby incorporated byreference herein in its entirety, including any figures, tables, ordrawings.

GOVERNMENT SUPPORT

This invention was made with government support under Grant NumberN00014-09-1-1008 awarded by the Office of Naval Research. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

MicroRNAs (miRNAs), typically 18 to 25 nt in length, arenon-protein-coding RNAs that can inhibit the translation of target mRNAs(Croce and Calin. 2005. miRNAs, cancer, and stem cell division. Cell122(1): 6-7). miRNAs directly or indirectly regulate a wide range ofgenes, and are involved in a remarkable spectrum of biological pathwaysincluding cell development, proliferation and apoptosis (He and Hannon.2004. MicroRNAs: small RNAs with a big role in gene regulation. Nat RevGenet 5(7): 522-31, Alvarez-Garcia and Miska. 2005. MicroRNA functionsin animal development and human disease. Development 132(21): 4653-62).As of September 2009, 10883 miRNA entries from vertebrates, flies,worms, plants, and viruses, including 721 human miRNAs and 579 mousemiRNAs, have been annotated (miRBase, Release 14) in the SangerInstitute miRNA sequence database (Griffiths-Jones, Saini, van Dongenand Enright. 2008. miRBase: tools for microRNA genomics. Nucleic AcidsRes 36(Database issue): D154-8); the function of many miRNAs is unknown.

Materials and methods that can detect and quantify miRNAs with highsensitivity and specificity are useful. Cellular miRNA profiles canoffer insights into gene expression, and allow the determination of thespecies, tissue types, and developmental stages of tissue samples.Further, the detection and quantification of miRNAs can lead to thediscovery of novel, miRNA-based diagnostic/prognostic biomarkers andtherapeutic agents.

However, detection of mature miRNAs is difficult due to several reasons.First, miRNAs are difficult to detect because they are relatively shortnucleic acid molecules (on average, only about 22 bases in length). Inaddition, these short sequences can be present in sequences other thanmature miRNA, such as pre-miRNA, pri-miRNA, genomic DNA and mRNA.Further, it is difficult to distinguish miRNAs within the same family,as these miRNAs usually differ from each other only in terms of one or afew nucleotides. Moreover, the melting temperatures (Tm) of miRNAs canvary greatly, from about 55° C. to 90° C.

At present, although a wide spectrum of miRNA detection techniques havebeen developed, there is a lack of high-throughput profiling assays thatcan sensitively and specifically detect miRNAs. Conventional techniquesfor miRNA profiling include Northern hybridization, cloning, andmicroarray analysis. (Wang, Ach and Curry. 2007. Direct and sensitivemiRNA profiling from low-input total RNA. RNA 13(1): 151-9, Wang andCheng. 2008. A simple method for profiling miRNA expression. Methods MolBiol 414: 183-90, Shingara, Keiger, Shelton, Laosinchai-Wolf, Powers,Conrad, Brown and Labourier. 2005. An optimized isolation and labelingplatform for accurate microRNA expression profiling. RNA 11(9): 1461-70,Nelson, Baldwin, Scearce, Oberholtzer, Tobias and Mourelatos. 2004.Microarray-based, high-throughput gene expression profiling ofmicroRNAs. Nat Methods 1(2): 155-61). These techniques are not assensitive or specific, when compared to quantitative real-time reversetranscription PCR (qRT-PCR).

Several qRT-PCR-based methods have been developed for detecting andquantifying miRNAs (Li, Yao, Huang, Wang, Sun, Fan, Chang, Li, Wang andXi. 2009. Real-time polymerase chain reaction microRNA detection basedon enzymatic stem-loop probes ligation. Anal Chem 81(13): 5446-51,Varkonyi-Gasic, Wu, Wood, Walton and Hellens. 2007. Protocol: a highlysensitive RT-PCR method for detection and quantification of microRNAs.Plant Methods 3: 12, Ro, Park, Jin, Sanders and Yan. 2006. A PCR-basedmethod for detection and quantification of small RNAs. Biochem BiophysRes Commun 351(3): 756-63). The current reverse transcriptasequantitative polymerase chain reaction assays (RT-qPCR), which use SYBRGreen, are lacking in specificity and sensitivity, as SYBR Green detectsall forms of nucleic acids, including double-stranded DNA,double-stranded RNA, single-stranded RNA and DNA, although the detectionsensitivity of double-stranded RNA, single-stranded RNA and DNA is lowerthan that of double-stranded DNA.

The most frequently used qRT-PCR-based method, developed by Chen et al.(Chen, Ridzon, Broomer, Zhou, Lee, Nguyen, Barbisin, Xu, Mahuvakar,Andersen, Lao, Livak and Guegler. 2005. Real-time quantification ofmicroRNAs by stem-loop RT-PCR. Nucleic Acids Res 33(20): e179), includestwo main steps: reverse transcription of miRNAs using stem-loop RTprimers, followed by a TaqMan® PCR analysis.

However, the Chen et al. method has several limitations. According toChen et al., profiling each target miRNA requires a target-specificTaqMan® probe and a target-specific RT primer. As a result, the cost ofmaking hundreds of target-specific probes and RT primers during miRNAscreening tests can be prohibitive. In addition, the Chen et al. methodis procedurally complex. Profiling each miRNA requires an RT reaction;otherwise, if only one RT reaction is performed, all miRNA-specific RTprimers need to be mixed together. Further, hundreds of target-miRNAspecific TaqMan® probes need to be added separately in order to detector quantify miRNA. Moreover, RT primers used in the Chen et al. methodonly have a 6-nt-sequence that base-pairs with the target miRNAs. As aresult, the RT primers may hybridize to, and prime, other RNAs duringthe RT reaction (Tang, Hajkova, Barton, Lao and Surani. 2006. MicroRNAexpression profiling of single whole embryonic stem cells. Nucleic AcidsRes 34(2):e9). Accordingly, improved methods for profiling miRNAs areneeded.

BRIEF SUMMARY OF THE INVENTION

The present invention provides materials and methods for detecting,quantifying, and/or profiling microRNAs. Advantageously, the presentinvention is sensitive, specific, convenient, and cost-effective.Further, the present invention can detect mature miRNA in a sample thatcomprises non-mature miRNA nucleic acids (such as one or more of genomicDNA, pre-miRNA, pri-miRNA and mRNA, and cDNAs thereof) that alsocomprise the target miRNA sequence.

In one aspect, materials for detecting, quantifying, and/or profilingmicroRNAs comprise: a universal primer for reverse transcription ofmiRNAs, a universal reverse primer for PCR amplification reaction, and auniversal probe (multiple universal probes can be used to increase thedetection sensitivity). Also provided are reagents and kits fordetecting, quantifying, and/or profiling miRNAs.

In some embodiments, the universal primer for reverse transcription isan oligonucleotide comprising: a (dT)_(n) sequence flanked by astem-looped universal adaptor sequence, wherein “n” is an integerranging from 8 to 50, wherein the universal primer comprises at leasttwo nucleotides adjacent to the 3′ end of the (dT)_(n) sequence, and thenucleotide immediately adjacent to the (dT)_(n) sequence is not T, andwherein the universal adaptor sequence near the 5′ end of the (dT)_(n)sequence forms a stem-loop structure by base-pairing.

In one embodiment, the universal reverse primer is an oligonucleotidecomprising a sequence that is, or base-pairs with, at least part of theadaptor sequence near the 5′ end of the (dT)_(n) sequence. In oneembodiment, the universal probe comprises a sequence that is, orbase-pairs with, at least part of the adaptor sequence near the 5′ endof the (dT)_(n) sequence.

In some embodiments, the universal primer for reverse transcriptioncomprises SEQ ID NO: 1. In some embodiments, the universal reverseprimer for PCR amplification comprises SEQ ID NO: 2. In someembodiments, the universal probe comprises SEQ ID NO: 3.

In another aspect, the present invention provides assays for detecting,quantifying, and/or profiling miRNAs. In one embodiment, the presentinvention can detect a plurality of target miRNAs using one reversetranscription reaction and one qPCR reaction.

Advantageously, the present invention can detect, quantify, and/orprofile miRNAs at a level of about 1 pg of total RNA.

In one embodiment, the method for detecting, quantifying, and/orprofiling a target miRNA comprises:

a) contacting a sample comprising miRNAs with an effective amount ofpoly(A)polymerase molecules to yield 3′ end-polyadenylated miRNAmolecules;

b) contacting the sample with an effective amount of a universal primerfor reverse transcription and reverse transcriptases, and reversetranscribing the polyadenylated miRNA molecules to yield correspondingc-DNA molecules; and

-   -   c) contacting the sample with an effective amount of a universal        reverse primer and a forward primer, and amplifying the        corresponding c-DNA molecules using an amplification reaction        (e.g., PCR).

In a further embodiment, the present invention uses Surveyor nucleaseand/or single strand endonucleases that remove mis-matches caused bymispriming to enhance specificity and/or single strand endonucleasessuch as Exonuclease I that remove single-stranded DNA such as excessprimers. In another embodiment, the present invention uses uracil-DNAGlycosylase (UDG) and/or dUTP to enhance specificity.

In some embodiments, a plurality of probes is used for detection and/orquantification of target miRNAs. In some embodiments, the universalprobe comprises one or more locked-nucleic acids (LNAs).

In one embodiment, the forward primer comprises the target-miRNAsequence, one or more additional nucleotides (such as adenine molecules)attached to the 3′ end of the target mature miRNA sequence, and one ormore additional nucleotides attached to the 5′ end of the target maturemiRNA sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of an embodiment of the qRT-PCR assayfor profiling miRNA, using a universal TaqMan® probe and a universal RTprimer. Briefly, miRNAs are polyadenylated using poly(A)polymerasemolecules. The poly(A)-tailed miRNAs are then reverse-transcribed intocDNAs using a universal primer comprising oligo dTs flanked by astem-loop adaptor sequence. Finally, the cDNAs are amplified by qRT-PCRsusing a mature target miRNA sequence as the forward primer and auniversal reverse primer (QRTU). In one embodiment, a universal TaqMan®probe is used to detect the amplification product. Q=Quencher, such asIABlk_FQ (Iowa Black™ FQ), F=Fluorescent Dye, such as FAM(6-carboxyfluorescein). SEQ ID NOS: 27-30 are sequences shown in FIG. 1.

FIGS. 2A-D show characteristics of the universal probe and RT primer(UPR) miRNA real-time quantitative PCR (qRT-PCR) assay. (FIG. 2A)Amplification curve plot of miR-690 UQmiR qRT-PCR assay. (FIG. 2B)Relative standard curve plot of miR-690 UQmiR qRT-PCR assay calculatedover 10 serial dilutions. Average C_(T) values (y-axis) are plottedagainst the logarithm of the input amount of RNA (x-axis) added to eachsample. Standard curve of the slope was −3.4 and correlation coefficientwas 0.9995. (FIG. 2C) Correlation of total RNA input to the threshold ofcycle (CT) values in four miRNA assays. Mouse spleen total RNA inputranged from 10 ng to 0.1 pg per RT reaction. (FIG. 2D) Amplificationcurve plot of 96 miRNA amplicons in a UQmiR qRT-PCR assay. Theexperiments were conducted in triplicate. During QPCR, a forward primerspecific to each target miRNA and a universal reverse primer (QRTU) wereused.

FIGS. 3A-D show that the UPR Q-RT-PCR assay is miRNA-specific. (FIG. 3A)Amplification curve plot of the UPR miRNA qRT-PCR assay, using total RNAas the template for reverse transcription without the step ofpolyadenylation (mRNA). When mRNAs are used as templates foramplification of let-7, miR-21, miR-142, miR-150 and miR-494, the cyclethreshold values (Cts) of qRT-PCRs are greater than 38. NTCs(nontemplate controls) did not produce any detectable signals. The Il-4and Il-18 amplifications of the cDNA molecules serve as positivecontrols of the RT reaction. (FIG. 3B) Amplification curve plot of theUPR miRNA qRT-PCR assay for let-7, miR-21, miR-142, miR-150, miR-494 andmiR-690. Mouse tail genomic DNA (DNA) and transcribed spleen total RNA(miRNA, served as positive controls) were subjected to polyadenylationand reverse transcription reactions. There are no detectable signals forthe DNA template. (FIG. 3C) Amplification curve plot of UPR miRNAqRT-PCR assay using mouse tail genomic DNA as the template of the 96miRNA qPCR array. (FIG. 3D) Agarose gel image of the 12 miRNA reactionsfrom the 96-miRNA qPCR array assay. After 40 cycles, products wereelectrophoresed on 2% agarose gels. A 1 kb DNA marker (Invitrogen) wasloaded on the left side of the gel.

FIGS. 4A-B show characteristics of the SYBR Green real-time quantitativePCR (qRT-PCR) assay for 96 miRNAs. The SYBR Green Q-PCRs were performedusing SYBR Green. For each miRNA, a forward primer specific to thetarget miRNA and a universal reverse primer were used for qPCRamplification. (FIG. 4A) Dissociation curves of all 96 miRNA ampliconsamplified using miRNA cDNAs prepared from mouse spleens. (FIG. 4B)Amplification curve plot of 96 miRNA amplicons of the SYBR Green Q-PCRassay. The experiments were conducted in triplicate.

FIG. 5 is a schematic illustration of an embodiment of the UQmiRquantitative real-time PCR assay for profiling miRNAs, using one or moreuniversal hydrolysis probes and a universal RT primer. Briefly, miRNAsare polyadenylated using poly(A)polymerase molecules. The poly(A)-tailedmiRNAs are then reverse-transcribed into cDNAs using a universal primercomprising oligo-dTs flanked by a stem-loop adaptor sequence (see Table2). Finally, the cDNAs are amplified by RT-qPCR using a UQmiR forwardprimer comprising the target mature miRNA sequence and a universalreverse primer (QRTU). Multiple universal hydrolysis probes can be usedto sensitively detect the amplicons. Q=quencher, (e.g., IABlk_FQ);F=fluorescent dye, (FAM).

FIG. 6 illustrates that the UQmiR miRNA RT-qPCR assay sensitively andspecifically amplifies mature miRNAs. In one embodiment, the forwardprimer (UQmiR primer) of the amplification reaction comprises the targetmature miRNA sequence and additional sequences attached to the 5′- and3′-ends of the mature miRNA sequence. The UQmiR-primer is annealed tothe cDNA of the target mature miRNA, and extension occurs at both endsof the primer-cDNA complex; however, unlike the complex formed by theUQmiR-primer and the mature miRNA cDNA, there is no extension at bothends of the complex formed by the UQmiR-primer and other DNA molecules.Attachment of additional sequences at the 5′- and 3′ ends of the targetmature miRNA sequence also results in higher Tm for the UQmiR primer.After extension reaction by a DNA polymerase, the Tm of the cDNAtemplate from a specific miRNA may be increased. Advantageously, theUQmiR primer does not anneal to, or has a low probability of annealingto, the nucleic acid molecules other than cDNA from mature miRNA (suchas, genomic DNA, cDNAs of pre-miRNA, pri-miRNA and mRNA) that containthe mature miRNA sequence; Due to lower homology between the UQmiRprimer and those molecules, the complex formed by them is less stablethan the complex formed by the UQmiR primer and the cDNA from specificmature miRNA.

FIG. 7 compares the sensitivity of the RT-qPCR reaction between theUQmiR assay of the present invention and the commercially-availablemiRNA RT-qPCR Taqman assay (Applied Biosystems (ABI)). Same amounts ofsynthetic miR-142 and Let-7a mature miRNAs were used for both miRNART-qPCR assays following the manufacturer's instruction. The resultsshow that the UQmiR assay of the present invention is at least two timesmore sensitive than the commercially-available Taqman assay.

FIGS. 8A-B show that the UQmiR miRNA qPCR assay of the present inventiondiscriminates mature miRNAs from pre-miRNAs. Mature miRNA-specific andpre-miRNA-specific PCR primers, which have extra sequences at the3′-ends but no extra sequences at the 5′-ends, were used to detectpre-miRNA. A total of 7.85E8 copies of synthetic pre-miR-142 andpre-miR-150 were added to the RT reaction. The results show that maturemiRNA-specific primers do not amply pre-miRNAs. The folds ofdiscrimination are over 50 times. The Y axis represents fluorescenceintensity corresponding to the specific amplification levels of thetemplate.

FIGS. 9A-C show that the UQmiR miRNA qPCR assay of the present inventiondiscriminates mature miRNAs from pre-miRNAs. UQmiR primers consisting ofsequences from mature miRNA and extra sequences that flank the maturemiRNA sequences were used to detect mature miRNA and pre-miRNAsequences. A total of 7.85E8 copies of synthetic Let-7a, miR-142,pre-Let-7a, pre-miR-142 and pre-miR-150 were added to the RT reaction.The results show that mature miRNA-specific primers do not amplypre-miRNAs. The folds of discrimination are over one thousand times.

FIG. 10 shows that the UQmiR miRNA qPCR assay of the present inventiondiscriminates mouse Let-7 miRNA family members. Four closely-relatedlet-7 family members (let-7a to let-7d) were used in the assay. RelativemiRNA detection percentages (%) to the perfectly matched targets werecalculated based on Cq values. A total of 7.85E8 copies of synthetic RNAwere added to the RT reaction. The numbers in parentheses indicate thedifference in bases between each pair of miRNAs.

FIG. 11 shows relative plasma miRNA expression levels of human plasmamiRNAs pooled from five normal volunteers measured by the RT-qPCR miRNAassay of the present invention. The results show that the presentRT-qPCR miRNA assay can sensitively and specifically detect 88 out of 94miRNAs (those miRNAs have high expression levels in mouse spleens fromthe dot blot array results) in a sample that only contains 0.8 ml ofplasma for each miRNA.

FIG. 12 shows that the UQmiR miRNA qPCR assay of the present inventiondiscriminates mature miRNAs from pre-miRNAs. Mature miRNA-specificprimers were used to detect mature miRNA and pre-miRNA sequences. Atotal of 7.85E8 copies of synthetic pre-miR-142 and pre-miR-150 wereadded to the RT reaction. The results show that mature miRNA-specificprimers do not amply pre-miRNA. The folds of discrimination are over onethousand times.

FIG. 13 shows discrimination of mature miRNA and pre-miRNA using theUQmiR miRNA qPCR assay with Surveyor nuclease, Exonuclease I nucleaseand UQmiR primer that only has 3′-end extra sequence. UQmiR primerscontaining mature miRNA sequences were used to detect mature miRNA andpre-miRNA sequences. A total of 7.85E8 copies of synthetic Let-7a,miR-142, pre-Let-7a, pre-miR-142 and pre-miR-150 were added to RTreaction. The results show that UQmiR primer for detecting mature miRNAprimers do not detect pre-miRNA well. The folds of discrimination areover one thousand times, which are shown above the each bar.

FIG. 14 shows discrimination UDG assay of mouse Let-7 miRNA familymembers. Four closely related let-7 family members (from let-7a tolet-7d) were included in the assay. Relative miRNA detection percentages(%) to the perfectly matched targets were calculated based on Cq values.A total of 7.85E8 copies of synthetic RNA were added to the RT reaction.The numbers in parentheses indicate different bases between each pair ofthe miRNAs. RT primer that contains multiple Uracil nucleotides wereused (LUTVN RT primer and Ublocker in Table 2), and after the doublestrand cDNA synthesis, a mixture of Uracil DNA glycosylase (UDG) and theDNA glycosylase-lyase Endonuclease VIII was used to remove LUTVN RTprimer and Ublocker. Very low levels of non-specific signal wereobserved, ranging from 0 to 0.18% (0 to 0.3, Chen et al. method) formiRNAs with 2-5 mismatched bases and only 0.11 to 0.88% (0.1 to 3.7,Chen et al. method) for the miRNAs that differed by a single nucleotide.

FIG. 15 illustrates an embodiment of UQmiR primers designed for theLet-7 miRNA family. UQmiR primers are designed so that they have minimalhomology between each other, especially within a miRNA family, toincrease UQmiR specificity between homologous miRNA sequences. Thenumber of different bases between each miRNA sequence is greatlyincreased from one or a few. The primers in FIG. 15 are mmu-let-7a (SEQID NO:19); mmu-let-7c (SEQ ID NO:20); mmu-let-7b (SEQ ID NO:21);mmu-let-7d (SEQ ID NO:22); mmu-let-7e (SEQ ID NO:23); mmu-let-7f (SEQ IDNO:24); mmu-let-7g (SEQ ID NO:25); and mmu-let-7i (SEQ ID NO:26).

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is a universal primer sequence for reverse transcription ofmiRNAs (RTUloop).

SEQ ID NO: 2 is a universal reverse primer sequence in qPCR reaction(QRTU).

SEQ ID NO: 3 is a universal probe sequence (miRU probe).

SEQ ID NO: 4 is a forward primer sequence for qPCR reaction (Let-7a).

SEQ ID NO: 5 is a forward primer sequence for qPCR reaction (miR-21).

SEQ ID NO: 6 is a forward primer sequence for qPCR reaction (miR-142).

SEQ ID NO: 7 is a forward primer sequence for qPCR reaction (miR-150).

SEQ ID NO: 8 is a forward primer sequence for qPCR reaction (miR-494).

SEQ ID NO: 9 is a forward primer sequence for qPCR reaction(mmu-miR-690).

SEQ ID NO: 10 is a universal primer sequence for reverse transcriptionof miRNAs.

SEQ ID NO: 11 is a universal reverse primer sequence in qPCR reaction.

SEQ ID NO: 12 is a Ublocker sequence useful according to the presentinvention.

SEQ ID NO: 13 is a universal primer sequence for reverse transcriptionof miRNAs.

SEQ ID NO: 14 is a universal reverse primer sequence in qPCR reaction.

SEQ ID NO: 15 is a universal reverse primer sequence in qPCR reaction.

SEQ ID NO: 16 is a universal hydrolysis LNA probe sequence.

SEQ ID NO: 17 is a universal hydrolysis LNA probe sequence.

SEQ ID NO: 18 is a universal hydrolysis LNA probe sequence.

SEQ ID NO: 19 is a primer sequence designed for the mmu-let-7a miRNAuseful according to the present invention (FIG. 15).

SEQ ID NO: 20 is a primer sequence designed for the mmu-let-7c miRNAuseful according to the present invention (FIG. 15).

SEQ ID NO: 21 is a primer sequence designed for the mmu-let-7b miRNAuseful according to the present invention (FIG. 15).

SEQ ID NO: 22 is a primer sequence designed for the mmu-let-7d miRNAuseful according to the present invention (FIG. 15).

SEQ ID NO: 23 is a primer sequence designed for the mmu-let-7e miRNAuseful according to the present invention (FIG. 15).

SEQ ID NO: 24 is a primer sequence designed for the mmu-let-7f miRNAuseful according to the present invention (FIG. 15).

SEQ ID NO: 25 is a primer sequence designed for the mmu-let-7g miRNAuseful according to the present invention (FIG. 15).

SEQ ID NO: 26 is a primer sequence designed for the mmu-let-7i miRNAuseful according to the present invention (FIG. 15).

SEQ ID NO: 27 is a poly(A) sequence (FIG. 1).

SEQ ID NO: 28 is a poly(T) cDNA sequence reverse-transcribed from apoly(A) sequence (FIG. 1).

SEQ ID NO: 29 is a poly(A) sequence (FIG. 1).

SEQ ID NO: 30 is a poly(A) sequence (FIG. 1).

DETAILED DISCLOSURE OF THE INVENTION

The present invention provides materials and methods for detecting,quantifying, and/or profiling microRNAs. The present invention uses areverse transcription reaction and a PCR amplification reaction. In oneembodiment, the present invention uses a universal probe, such as aTaqMan® probe, and a universal RT-primer (UPR).

Advantageously, the UQmiR qRT-PCR assay can be sensitive, specific,convenient, and cost-effective. In one embodiment, the present inventionuses a universal probe (such as a TaqMan®) and a universal RT primer(UPR). In a preferred embodiment, the present invention can detect aplurality of target miRNAs using one RT reaction and a single universalprobe (multiple universal probes may be used to increase the detectionsensitivity). Advantageously, the present invention allows detection andquantification of miRNAs in as little as 1 pg total RNA. Further, thepresent invention can detect mature miRNA in a sample that containnon-mature miRNA nucleic acids (such as genomic DNA, pre-miRNA,pri-miRNA and mRNA, and cDNAs thereof) that also comprise the targetmiRNA sequence. For instance, genomic DNA and mRNA in total RNA samplesproduce no or little detectable signals.

Total RNA used in the present invention can be obtained from simpleextraction methods, such as, Trizol extraction. Total RNA samples usedin the present invention need not be treated with DNases or undergosmall RNA fractionation or purification, which are not only laborintensive procedures, but also may result in significant loss of inputmiRNAs.

The miRNA UQmiR qRT-PCR assay of the present invention has two majoradvantages when compared to the conventional miRNA stem-loop qRT-PCRmethods (such as the Chen et al. method). First, a highly specificuniversal poly (T) primer, for example with a stretch of 25 Ts, is usedto prime the RT reaction for detection of all target miRNAs. Incontrast, the conventional methods use a stem-loop RT primer that has a6 nt sequence specific to each target miRNA sequence. For example, the25 Ts poly (T) sequence in the universal primer theoretically appearsonly once in a random sequence of 1.1259E+15 bps, while the 6nt-sequence in the miRNA-specific primer appears 652962 times in arandom sequence of the mouse genomic size. Further, the 6 nt,target-miRNA-specific primer used in the prior art methods is notgenome-wide specific; It can not only prime the target miRNAs, but alsoprime other RNAs that have the 6 nt sequences. Further, using the priorart primers requires lower temperature (16° C.) for RT reaction (Chen,Ridzon, Broomer, Zhou, Lee, Nguyen, Barbisin, Xu, Mahuvakar, Andersen,Lao, Livak and Guegler. 2005. Real-time quantification of microRNAs bystem-loop RT-PCR. Nucleic Acids Res 33(20): e179). Therefore, thepresent invention can greatly increase the priming specificity duringreverse transcription, since only RNAs that have poly (A) tails can bereversely transcribed. The decrease of non-specificity in the RTreaction increases the sensitivity of the qRT-PCR assay.

Moreover, the present invention can use a universal probe (such as auniversal TaqMan® probe) for the detection and qualification of aplurality of miRNAs. In contrast, the conventional methods requiretarget-miRNA-specific TaqMan® probes, where each probe can only detectone target miRNA. In addition, conventional methods require that eachtarget-specific probe need to be individually added each time. Thepresent invention also embodies the use of multiple universal probes toincrease the detection sensitivity.

In a further embodiment, the present invention provides a RT-qPCR miRNATaqman assay (UQmiR) for profiling miRNAs. In one embodiment, thepresent invention also involves use of computer programs (such as QmiR)for detection, quantification and/or profiling of miRNAs. In oneembodiment, the RT-qPCR miRNA assay comprises one RT reaction with oneuniversal RT primer, one universal reverse primer for the amplificationreaction, miRNA-specific UQmiR forward primers, and one or moreuniversal hydrolysis probes to sensitively detect all miRNAs (FIG. 5 andTable 2). The UQmiR forward primers only detect mature miRNA sequences,and do not amplify non mature miRNA sequences, such as genomic DNA,cDNAs of pre-miRNA, pri-miRNA and mRNA (FIG. 6).

In one embodiment, the present invention also comprises the use of acomputer program, such as the QmiR computer program, to enhancespecificity of the RT-qPCR miRNA Taqman assay (UQmiR) assay in thedetection, quantification and/or profiling of miRNAs.

In a further embodiment, the present invention uses Surveyor nucleasethat remove mis-matches caused by mispriming to enhance specificityand/or single strand endonucleases such as Exonuclease I that removesingle strand DNA such as excess primers. In another embodiment, thepresent invention uses uracil-DNA Glycosylase (UDG) and/or dUTP toenhance specificity.

In one embodiment, UDG can be used to destroy the templates of anynon-specific amplification when use dUTP-containing oligos as RTprimers. FIGS. 13-15 describe various embodiments of using Surveyornuclease, endonucleases, uracil-DNA Glycosylase (UDG) and/or dUTP forenhancing specificity and sensitivity.

Materials for miRNA Profiling

One aspect of the present invention provides materials for detecting,quantifying, and/or profiling miRNAs. In one embodiment, the materialscomprise: a universal primer for reverse transcription of miRNAs, auniversal reverse primer for PCR amplification reaction, and a universalprobe. Also provided are reagents and kits for detecting, quantifying,and/or profiling miRNAs.

Design of Universal Primers for Reverse Transcription of miRNAs

In one aspect, the present invention provides a universal primer forreverse transcription of miRNAs. In some embodiments, the universalprimer is an oligonucleotide sequence comprising: a (dT)_(n) sequenceflanked by a stem-looped universal adaptor sequence, wherein n is aninteger ranges from 8 to 50, wherein the universal primer comprises atleast two nucleotides adjacent to the 3′ end of the (dT)_(n) sequence,and the nucleotide immediately adjacent to the (dT)_(n) sequence is notT, and wherein the universal adaptor sequence near the 5′ end of the(dT)_(n) sequence forms into a stem-loop structure by base-pairing. Inone embodiment, the universal primer for reverse transcription issingle-stranded DNA.

In some embodiments, n is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In someembodiments, n can be an integer less than 10 or greater than 50.

In some embodiments, the universal primer comprises at least 2, 3, 4, 5,6, 7, 8, 9, or 10 nucleotides are immediately adjacent to the 3′ end ofthe (dT)_(n) sequence.

In some embodiments, the adaptor sequence comprises 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 56, 57, 58, 59, or 60nucleotides. In some embodiments, the adaptor sequence comprises morethan 60 nucleotides. In some embodiments, each stem of the adaptorsequence located near the 5′ end of the (dT)_(n) sequence comprises 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or25 nucleotides. In one embodiment, the stems of the adaptor sequencesbase pair with each other (see FIGS. 1 and 5). In some embodiments, eachstem of the adaptor sequence located near the 5′ end of the (dT)_(n)sequence comprises more than 25 nucleotides. In some embodiments, theloop of the adaptor sequence comprises 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In certain embodiments,the loop of the adaptor sequence can comprise more than 25 nucleotides.In one embodiment, the loop of the adaptor comprises single-strandednucleotides.

FIGS. 1 and 5 illustrate embodiments of the universal primer for reversetranscription of miRNAs. In a specific embodiment, the universal primerfor reverse transcription of miRNAs comprises SEQ ID NO: 1. In somespecific embodiments, the universal primer for reverse transcription ofmiRNAs comprises SEQ ID NO: 10 or SEQ ID NO: 13.

In some embodiments, the adaptor sequence (near the ′5 end) of theuniversal primer for reverse transcription of miRNAs does not comprise asequence that hybridizes, or base-pairs, with the target miRNA, whereinthe sequence is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 20 nucleotides in length. In one embodiment, the universalprimer for reverse-transcription is not target miRNA specific.

Design of Universal Reverse Primers for PCR Amplification Reaction

In one aspect, the present invention provides a universal reverse primerfor PCR amplification reaction. In some embodiments, the universalreverse primer is an oligonucleotide comprising a sequence that is, orbase-pairs with, at least part of the adaptor sequence (located near the5′ end of the (dT)_(n) sequence) of the universal primer for reversetranscription. In some embodiments, the universal reverse primer is nottarget miRNA specific. In some embodiments, the universal reverse primeris single-stranded DNA.

In some embodiments, the universal reverse primer for PCR amplificationcomprises 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, or 25 nucleotides. In some embodiments, the universal reverse primerfor PCR amplification comprises less than 8 or more than 25 nucleotides.

FIGS. 1 and 5 illustrate embodiments of the universal reverse primer forPCR amplification reaction. In a specific embodiment, the universalreverse primer for PCR amplification reaction comprises SEQ ID NO: 2.

In certain embodiments, the universal reverse primer for PCRamplification does not comprise a sequence that is, or base-pairs with,the target miRNA sequence. In one embodiment, the universal reverseprimer for PCR amplification is not target miRNA specific.

In one embodiment, the universal reverse primer for PCR amplificationcomprises one or more dUTPs. In some embodiments, the universal reverseprimer for PCR amplification reaction comprises SEQ ID NO: 11, SEQ IDNO: 14, or SEQ ID NO: 15.

Design of Forward Primers for PCR Amplification Reaction

In one aspect, the present invention provides a mature miRNA-specificforward primer for PCR amplification reaction. In one embodiment, theforward primer is miRNA-specific. In some embodiments, the forwardprimer comprises at least part of the target miRNA sequence. In someembodiments, the forward primer comprises the entire target miRNAsequence. In some embodiments, the forward primer is single-strandedDNA.

In some embodiments, the forward primer comprises at least 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25consecutive nucleotides of the target miRNA sequence. In one embodiment,the forward primer comprises one or more additional nucleotides (such asadenine molecules) attached to the 3′ end of the target mature miRNAsequence. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides(such as adenine molecules) are attached to the 3′ end of the targetmature miRNA sequence. By attaching the additional nucleotides to the 3′end of the target miRNA sequence, the forward primer can detect thetarget miRNA sequence other than non mature miRNA sequences (e.g.,genomic DNA and cDNAs of pre-miRNA, pri-miRNA) with higher specificity.As shown in FIG. 6, cDNAs of pre-miRNA, pri-miRNA and mRNA sequences andgenomic DNA sequences do not extend at their 3′-end although they mayanneal to the UQmiR forward primer comprising the additional nucleotides(such as adenine molecules).

In one embodiment, the forward primer comprises one or more additionalnucleotides attached to the 5′ end of the target mature miRNA sequence.In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 additionalnucleotides are attached to the 5′ end of the target mature miRNAsequence.

The number of nucleotides attached to the 3′ and 5′ ends of the forwardprimer can be adjusted to achieve the desired Tm of the forward primer.In some embodiments, Tm is about 60° C. to 75° C., 60° C. to 72° C., 65°C. to 72° C., 67° C. to 72° C., or 69° C. to 71° C. In some embodiments,Tm is about 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., 71° C., 72°C., 73° C., 74° C., and/or 75° C. Preferably, Tm is about primer 70° C.In some embodiments, variation of Tm of the forward primer is within0.5° C., 1° C., 1.5° C., 2° C., 2.5° C., 3° C., 3.5° C., 4° C., 4.5° C.,5° C., 5.5° C., 6° C., 6.5° C., 7° C., 8° C., 9° C., or 10° C. FIGS. 1,5 and 6 illustrate embodiments of the forward primer for PCRamplification reaction.

Design of Universal Probes

In one aspect, the present invention provides a universal probe. Theuniversal probes are useful for the detection and/or quantification oftarget miRNAs. In some embodiments, the universal probe is anoligonucleotide comprising a sequence that is, or base-pairs with, atleast part of the adaptor sequence of the universal primer for reversetranscription. In some embodiments, the universal probe issingle-stranded DNA.

In some embodiments, the universal probe further comprises afluorophore, or other detectable moiety, attached at the ends of theoligonucleotide.

In certain embodiments, the universal probe in PCR amplificationcomprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or 25 nucleotides. In certain embodiments, theuniversal probe can comprise more than 25 nucleotides.

In some embodiments, a plurality of probes can be used for detectionand/or quantification of target miRNAs. In some embodiments, theuniversal probe comprises one or more locked-nucleic acids (LNAs).

In a specific embodiment, the universal probe comprises a sequence thatis, or base-pairs with, at least part of a stem-looped adaptor sequencenear the 5′ end of the (dT)n sequence of the adaptor sequence, whereinthe probe comprises one or more locked nucleic acids.

FIGS. 1 and 5 illustrate embodiments of the universal probe. In aspecific embodiment, the universal probe comprises SEQ ID NO: 3. In somespecific embodiments, the universal probe comprise SEQ ID NO: 16, 17 or18.

In certain embodiments, the universal probe does not comprise a sequencethat is, or base-pairs with, the target miRNA sequence. In oneembodiment, the universal probe is not target miRNA specific.

The detectable moiety is preferably a fluorophore. The fluorophores arepreferably attached to the ends or near the ends of the oligonucleotide.When a probe of the invention hybridizes with a target nucleic acidsequence, the probe undergoes a conformational change to bring thefluorophores closer in proximity to each other. This change in distancecauses a change in the photon absorption or emission of thefluorophores, creating a visual indication that the probe of theinvention has bound a target sequence.

Fluorescent resonance energy transfer (FRET) or non-FRET interactionsare used to detect the binding of the probe to its target sequence(e.g., PCR amplification products). FRET interactions (also known asnon-radiative energy transfer; see Yaron et al., Analytical Biochemistry95:228-235 (1979)) for quenching fluorescence signals requires spectraloverlap between the donor and acceptor fluorophore moieties and theefficiency of quenching is directly proportional to the distance betweenthe donor and acceptor moieties of the FRET pair. Extensive reviews ofthe FRET phenomenon are described in Clegg, R. M., Methods Enzymol.,221: 353-388 (1992) and Selvin, P. R., Methods Enzymol., 246: 300-334(1995). In contrast, non-FRET interactions (also known as radiationlessenergy transfer; See: Yaron et al., Analytical Biochemistry 95:228-235(1979)) requires short range interaction by “collision” or “contact”between the fluorophore moieties and therefore requires no spectraloverlap between the donor and acceptor pair.

When the probe binds to the target sequence, the probe will undergo aconformational change causing the distance and/or angle between thefluorophore pairs to change. This change can then be detected because itwill change the efficiency of resonance energy transfer between thefluorophore moieties after exposure of the probe to an excitationwave-length of light.

In one embodiment, fluorophores useful according to the presentinvention include, but are not limited to, FAM (6-carboxyfluorescein),CY5, CY3, BODIPY FL, and TEXAS RED. In a preferred embodiment, thefluorophore is FAM.

The universal primer, probe and adaptor sequences can be derived fromuniversal probe and primer sequences known in the art, such as universalTaqMan® probe and primer sequences. For example, the design of thestem-loop universal adaptor sequence is described in (Chen, Ridzon,Broomer, Zhou, Lee, Nguyen, Barbisin, Xu, Mahuvakar, Andersen, Lao,Livak and Guegler. 2005. Real-time quantification of microRNAs bystem-loop RT-PCR. Nucleic Acids Res 33(20): e179).

The oligonucleotides of the present invention can encompass single anddouble-stranded RNA, single and double-stranded DNA and cDNA, nucleicacid analogs, aptamers, and the like. The terms “nucleic acid” and“oligonucleotide” are used interchangeably herein. Preferably, theoligonucleotide strands of the probe are single-stranded DNA.

“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between a particular purine and a particular pyrimidine indouble-stranded nucleic acid molecules (DNA-DNA, DNA-RNA, or RNA-RNA).The major specific pairings are guanine with cytosine and adenine withthymine or uracil. Various degrees of stringency of hybridization can beemployed. The more severe the conditions, the greater thecomplementarity that is required for duplex formation. Severity ofconditions can be controlled by temperature, probe concentration, probelength, ionic strength, time, and the like.

Preferably, hybridization is conducted under high stringency conditionsby techniques well known in the art, as described, for example, inKeller, G. H. & M. M. Manak, DNA Probes, and the companion volume DNAProbes: Background, Applications, Procedures (various editions,including 2^(nd) Edition, Nature Publishing Group, 1993). Hybridizationis also described extensively in the Molecular Cloning manuals publishedby Cold Spring Harbor Laboratory Press, including Sambrook & Russell,Molecular Cloning: A Laboratory Manual (2001). Each of thesepublications is incorporated herein by reference in its entirety.

A non-limiting example of high stringency conditions for hybridizationis at least about 6×SSC and 1% SDS at 65° C., with a first wash for 10minutes at about 42° C. with about 20% (v/v) formamide in 0.1×SSC, andwith a subsequent wash with 0.2×SSC and 0.1% SDS at 65° C. Anon-limiting example of hybridization conditions are conditions selectedto be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, or 25° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence in the particular solution.T_(m) is the temperature (dependent upon ionic strength and pH) at which50% of the target sequence hybridizes to a perfectly matched probe.T_(m) typically increases with [Na⁺] concentration because the sodiumcations electrostatically shield the anionic phosphate groups of thenucleotides and minimize their repulsion. The washes employed may be forabout 5, 10, 15, 20, 25, 30, or more minutes each, and may be ofincreasing stringency if desired.

Calculations for estimating T_(m) are well-known in the art. Forexample, the melting temperature may be described by the followingformula (Beltz, G. A., K. A. Jacobs, T. H. Eickbush, P. T. Cherbas, andF. C. Kafatos, Methods of Enzymology, R. Wu, L. Grossman and K. Moldave[eds.] Academic Press, New York 100:266-285, 1983).

Tm=81.5° C.+16.6 Log [Na+]+0.41(% G+C)−0.61(% formamide)−600/length ofduplex in base pairs.

A more accurate estimation of T_(m) may be obtained usingnearest-neighbor models. Breslauer, et al., Proc. Natl. Acad. Sci. USA,83:3746-3750 (1986); SantaLucia, Proc. Natl Acad. Sci. USA, 95:1460-1465 (1998); Allawi & SantaLucia, Biochemistry 36:10581-94 (1997);Sugimoto et al., Nucleic Acids Res., 24:4501-4505 (1996). T_(m) may alsobe routinely measured by differential scanning calorimetry (Duguid etal., Biophys J, 71:3350-60, 1996) in a chosen solution, or by othermethods known in the art, such as UV-monitored melting. As thestringency of the hydridization conditions is increased, higher degreesof homology are obtained.

Kits

The present invention also provides kits for detecting, quantifying,and/or profiling miRNA. In one embodiment, the kit comprises a universalprimer for reverse transcription, a universal reverse primer for anamplification reaction (e.g., PCR), and/or a universal probe.Optionally, the kit can further comprise a forward primer sequence foran amplification reaction (e.g., PCR).

In a specific embodiment, the kit comprises SEQ ID NO:1 and/or SEQ IDNO:2. In another specific embodiment, the kit further comprises SEQ IDNO:3. In a further specific embodiment, the kit comprises one or more ofSEQ ID NOs: 4-9. In a specific embodiment, the kit comprises one or moreof SEQ ID NO:s 1-18.

Optionally, the kit may include any material useful for performing anystep of the present invention. For instance, the kit may furthercomprise any material useful for reverse transcription of RNAs, and/orfor profiling the target RNA. For instance, the kit maypoly(A)polymerases, dNTPs, Adenosine-5′-triphosphates (ATP), DNA ligases(e.g., T4 DNA ligase), and Taq DNA polymerase.

The kit may also comprise, e.g., a buffering agent, a preservative, or astabilizing agent. Each component of the kit is usually enclosed withinan individual container and all of the various containers are within asingle package along with instructions (e.g., printed instructions).

UQmiR qRT-PCR Assay

Another aspect of the present invention provides an assay for detecting,quantifying, and/or profiling miRNAs. Advantageously, the presentinvention can detect, quantify, and/or profile miRNAs in a sample ofabout 1 pg.

In one embodiment, the method for detecting, quantifying, and/orprofiling a target miRNA comprises:

a) contacting a sample containing miRNAs with an effective amount ofpoly(A)polymerase molecules to yield 3′ end-polyadenylated miRNAmolecules,

b) contacting the sample with an effective amount of a universal primerfor reverse transcription and reverse transcriptases, and reversetranscribing the polyadenylated miRNA molecules to yield correspondingc-DNA molecules, and

c) contacting the sample with an effective amount of a universal reverseprimer and a target miRNA-specific forward primer, and amplifying thecorresponding c-DNA molecules using an amplification reaction (e.g.,PCR);

wherein the universal primer for reverse transcription is anoligonucleotide comprising: a (dT)_(n) sequence flanked by a stem-loopeduniversal adaptor sequence, wherein n is an integer ranges from 8 to 50,wherein the universal primer comprises at least two nucleotides adjacentto the 3′ end of the (dT)_(n) sequence, and the nucleotide immediatelyadjacent to the (dT)_(n) sequence is not T, and wherein the universaladaptor sequence near the 5′ end of the (dT)_(n) sequence forms into astem-loop structure by base-pairing,

wherein the universal reverse primer for the amplification reaction isan oligonucleotide comprising a sequence that is, or base-pairs with, atleast part of the adaptor sequence of the universal primer for reversetranscription.

In one embodiment, the forward primer comprises the target-miRNAsequence, one or more additional nucleotides (such as adenine molecules)attached to the 3′ end of the target mature miRNA sequence. In someembodiments, 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides (such as adeninemolecules) are attached to the 3′ end of the target mature miRNAsequence. In one embodiment, the forward primer further comprises one ormore additional nucleotides attached to the 5′ end of the target maturemiRNA sequence. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, or 9additional nucleotides are attached to the 5′ end of the target maturemiRNA sequence.

In a further embodiment, the assay comprises the steps of:

contacting the sample with an effective amount of a universal probe,

detecting and/or quantifying a level of amplified c-DNA molecules, and

determining the level of the target miRNA based on the c-DNA level.

In one embodiment, a plurality of probes can be used for detectingand/or quantifying a level of amplified c-DNA molecules. In someembodiments, 2, 3, 4, 5, 6, or 7 probes are used for detecting and/orquantifying a level of amplified c-DNA molecules. In one embodiment, theuniversal probe comprises a detectable moiety, for example, afluorophore. In one embodiment, a plurality of detectable moieties(e.g., fluorophores) can be used.

In one embodiment, the amplification reaction (e.g., PCR) for amplifyingcDNAs is quantitative real time polymerase chain reaction (qRT-PCR).

In one embodiment, the PCR reaction is performed once. In anotherembodiment, the reverse transcription reaction is performed once.

In one embodiment, the miRNA profiling assay of the present inventionuses one universal primer for reverse transcription, one universalreverse primer in the amplification reaction (e.g., PCR), and/or oneuniversal probe. In one embodiment, the miRNA profiling assay detects,quantifies, and/or profiles a plurality of target miRNAs.

In one embodiment, the threshold cycle (Ct) of the PCR amplificationreaction ranges from 15 to 37. In preferred embodiments, the Ct value ofthe PCR amplification reaction ranges from 17 to 35, 20 to 30, 23 to 33,or 25 to 30. In certain embodiments, the Ct value is 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or37. In one embodiment, the Ct value does not exceed 38.

Alternatively, the UQmiR qRT-PCR assay can be used for detecting,quantifying, and/or profiling nucleotide molecules, such as for example,siRNAs, oligonucleotides, polynucleotides, and/or mRNAs.

In one embodiment, samples containing miRNAs is a total RNA sample. Incertain embodiments, the sample contains DNA, or has not been treatedwith DNases, or both. The samples can be derived from an organism,including mammals such as apes, chimpanzees, orangutans, humans,monkeys; and domesticated and/or laboratory animals such as dogs, cats,horses, cattle, pigs, sheep, goats, chickens, mice, rats, guinea pigs,and hamsters. The samples can be derived from, including but not limitedto, a sample containing tissues, cells, and/or biological fluidsisolated from a subject.

EXEMPLIFIED EMBODIMENTS Embodiment 1

A method for detecting, quantifying, and/or profiling a target miRNA,comprising:

a) contacting a sample containing miRNAs with an effective amount ofpoly(A)polymerase molecules to yield 3′ end-polyadenylated miRNAmolecules,

b) contacting the sample with an effective amount of a universal primerfor reverse transcription and reverse transcriptases, and reversetranscribing the polyadenylated miRNA molecules to yield correspondingc-DNA molecules; and

c) contacting the sample with an effective amount of a universal reverseprimer and a target miRNA-specific forward primer, and amplifying thecorresponding c-DNA molecules using an amplification reaction;

wherein the universal primer for reverse transcription is anoligonucleotide comprising: a (dT)_(n) sequence flanked by a stem-loopeduniversal adaptor sequence, wherein “n” is an integer ranging from 8 to50, wherein the universal primer comprises at least two nucleotidesadjacent to the 3′ end of the (dT)_(n) sequence, and the nucleotideimmediately adjacent to the (dT)_(n) sequence is not T, wherein theuniversal adaptor sequence near the 5′ end of the (dT)_(n) sequenceforms into a stem-loop structure by base-pairing, and

wherein the universal reverse primer in the amplification reaction is anoligonucleotide comprising a sequence that is, or base-pairs with, atleast part of the adaptor sequence near or toward the 5′ end of the(dT)_(n) sequence.

Embodiment 2

The method, according to embodiment 1, wherein the forward primercomprises the target miRNA sequence and three or more additionalnucleotides attached to the 3′ end of the target mature miRNA sequence.

Embodiment 3

The method, according to embodiment 1, wherein the additionalnucleotides attached to the 3′ end of the target mature miRNA sequenceare adenine molecules.

Embodiment 4

The method, according to embodiment 2, wherein one or more additionalnucleotides are attached to the 5′ end of the target mature miRNAsequence.

Embodiment 5

The method, according to any of the preceding embodiments, wherein theamplification reaction for cDNA amplification is quantitative real timepolymerase chain reaction (qRT-PCR).

Embodiment 6

The method, according to any one of embodiments 1-5, comprising:

contacting the sample with a sufficient amount of a universal probe,

detecting and/or quantifying a level of amplified c-DNA molecules, and

determining the level of the target miRNA based on the c-DNA level;

wherein the universal probe comprises a sequence that is, or base-pairswith, at least part of the adaptor sequence near the 5′ end of the(dT)_(n) sequence.

Embodiment 7

The method, according to embodiment 6, wherein the universal probecomprises locked nucleic acid (LNA) molecules.

Embodiment 8

The method, according to embodiment 6 or 7, wherein the universal probeincludes a detectable moiety.

Embodiment 9

The method, according to embodiment 8, wherein the detectable moiety isa fluorophore selected from FAM, CY5, CY3, BODIPY FL, TEXAS RED, or anycombination of two or more of the foregoing.

Embodiment 10

The method, according to any one of embodiments 6-9, wherein at leasttwo universal probes are used.

Embodiment 11

The method, according to any one of the preceding embodiments, whereinthe amplification reaction for amplification of cDNA molecules isperformed once.

Embodiment 12

The method, according to any one of the preceding embodiments, whereinthe threshold cycle (Ct) of the amplification reaction for amplificationcDNA molecules ranges from 17 to 35.

Embodiment 13

The method, according to any one of the preceding embodiments, whereinthe sample is a total RNA sample.

Embodiment 14

The method, according to any one of the preceding embodiments, whereinthe method is capable of detecting, quantifying and/or profilingmicroRNA in a total RNA sample of about 1 pg.

Embodiment 15

The method, according to any one of the preceding embodiments, whereinthe universal reverse primer comprises dUTP.

Embodiment 16

The method, according to embodiment 15, further comprising addinguracil-DNA Glycosylase molecules to the amplification reaction mixture.

Embodiment 17

The method, according to any one of the preceding embodiments,comprising adding nuclease molecules to the reaction mixture.

Embodiment 18

A universal primer for detecting, quantifying and/or profiling miRNA,wherein the universal primer is a primer for reverse transcription ofmiRNAs, wherein the universal primer is an oligonucleotide comprising: a(dT)_(n) sequence flanked by a stem-looped universal adaptor sequence,wherein “n” is an integer ranging from 8 to 50, wherein the universalprimer comprises at least two nucleotides adjacent to the 3′ end of the(dT)_(n) sequence, and the nucleotide immediately adjacent to the(dT)_(n) sequence is not T, and wherein the universal adaptor sequencenear the 5′ end of the (dT)_(n) sequence forms into a stem-loopstructure by base-pairing.

Embodiment 19

A universal primer for detecting, quantifying and/or profiling miRNA,wherein the universal primer is a reverse primer for an amplificationreaction, wherein the universal primer is an oligonucleotide comprisinga sequence that is, or base-pairs with, at least part of the adaptorsequence near the 5′ end of the (dT)_(n) sequence according toembodiment 18.

Embodiment 20

The universal reverse primer for an amplification reaction according toembodiment 19, wherein the universal reverse primer comprises dUTP.

Embodiment 21

A universal probe for detecting, quantifying and/or profiling miRNA,wherein the universal probe comprises a sequence that is, or base-pairswith, at least part of the adaptor sequence near the 5′ end of the(dT)_(n) sequence of embodiment 18.

Embodiment 22

The universal probe according to embodiment 21, further comprising adetectable moiety.

Embodiment 23

The universal probe according to embodiment 21 or 22, wherein thedetectable moiety comprises a fluorophore.

Embodiment 24

The universal probe according to any one of embodiments 21-23,comprising locked nucleic acid (LNA) molecules.

Embodiment 25

A kit for detecting, quantifying and/or profiling a level of expressionof miRNA, comprising a universal primer for reverse transcriptionaccording to embodiment 18; and a universal reverse primer for anamplification reaction according to embodiment 19 or 20.

Embodiment 26

The kit according to embodiment 25, further comprising a universal probeof any one of embodiments 21-24.

Materials and Methods

Table 1 illustrates oligonucleotide sequences useful according to thepresent invention.

TABLE 1 Name Sequence (5′ to 3′) RTUloopTGGCTAGTTAAGCTCACCAGCTCGGTACCAAGCTTAA CTAGCCA(T)25N*N** (SEQ ID NO: 1)QRTU TGGCTAGTTAAGCTCACCAGCTCG  (SEQ ID NO: 2) miRU probe FAM/TGGCTAGTTAAGCTTGGTACCGAGCT/ IABlk_FQ (SEQ ID NO: 3) Let-7aTGAGGTAGTAGGTTGTATAGTT (SEQ ID NO: 4) miR-21TAGCTTATCAGACTGATGTTGA (SEQ ID NO: 5) miR-142TGTAGTGTTTCCTACTTTATGGA (SEQ ID NO: 6) miR-150TCTCCCAACCCTTGTACCAGTG (SEQ ID NO: 7) miR-494 TGAAACATACACGGGAAACCTCTT (SEQ ID NO: 8) mmu-miR-690 AAAGGCTAGGCTCACAACCAAA (SEQ ID NO: 9) N* = A,G, or C; N** = A, G, C, or T. ABBREVIATIONS: miRNAs: MicroRNAs; UPR: auniversal probe and RT primer; qRT-PCR: quantitative real-time reversetranscription PCR; QRTU: universe reverse primer; RTUloop: universal RTprimer; FAM: 6-carboxyfluorescein; CT: threshold cycle.

TABLE 2 Name Sequence (5′ to 3′) Longshort RTCCATCAATCGTGTGTCTCTATGGATGCTGTCACAAC primerGACATGTCAGCCTCTGACTCCAGGATCTGTAGACGCTAGCTGACATGTCGTTGTGACAGCATCCATAGAGACACACGATTGATGGTTTTTTTTTTTTTTTTTTTTTTT TTN*N** (SEQ ID NO: 10) NewURPGCCTCTGACTCCAGGATCTGTAGAC  (SEQ ID NO: 11) UblockerCGACAUGUCAGCUAGGUCGUttCGGUttCACUAUCG CUACGCACAG (SEQ ID NO: 12) LUTVN RTCUGUGCGUAGCGAUAGUGAAACCGAAACGACCUAGC primer UGACAUGUCGTTTTTTTTN*N**(SEQ ID NO: 13) 384URP CTGTGCGTAGCGATAGTGAAACCGAAAC (SEQ ID NO: 14) URPCGCTGTACTCCAGGATCTGTAGACG (SEQ ID NO: 15) LNADLP1[6FAM]ACC[A][T][C]A[A][T][C]G[T]G[T] G[BHQ1]** (SEQ ID NO: 16) LNADLP2[6FAM]CT[A][T][G]G[A]T[G]C[T]G[T][C] A[BHQ1]** (SEQ ID NO: 17) LNADLP3[6FAM]C[G]A[C]AT[G][T][C]A[G][C][T] AG[BHQ1]** (SEQ ID NO: 18) N* = A,G, or C; N** = A, G, C, or T. ** The bases in parenthesis are specialbases called LNAs (locked nucleic acids).

EXAMPLES

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1 The Universal Probe-RT Primer (UPR) MicroRNA Real-Time qRT-PCRAssay

FIG. 1 illustrates one embodiment of the universal probe-RT primer (UPR)real-time qRT-PCR assay for profiling microRNAs. The assay comprises athree-step process. First, total RNA is polyadenylated usingpoly(A)polymerase molecules. The polyadenylated miRNAs are reverselytranscribed into cDNAs using a universal RT primer (RTUloop) with 25-Tpoly (T) at 3′ end. Finally, the RT product is amplified by qPCR usingthe mature target miRNA sequence as the forward primer and a universalreverse primer (QRTU). The amplification products can be detected orquantified using a miRU fluorescent probe, such as for example, a26-nucleotide universal TaqMan® probe.

MiRNA Sequences and Primers

About 800 human and mouse miRNA genes are selected from the SangerInstitute miRBase Sequence Database. miRNA expression profiling wasperformed and analyzed using the dot-blot array as previously described(Wang and Cheng. 2008. A simple method for profiling miRNA expression.Methods Mol Biol 414: 183-90). 96 miRNAs were selected for miRNA QPCRarray assay from the dot blot array results.

The miRNA universal TaqMan® probe was designed by PrimerQuest (Table 1)based on sequences described in Ro et al. The stem-loop adaptorcontained in the RT primers were designed according to Chen et al.(Chen, Ridzon, Broomer, Zhou, Lee, Nguyen, Barbisin, Xu, Mahuvakar,Andersen, Lao, Livak and Guegler. 2005. Real-time quantification ofmicroRNAs by stem-loop RT-PCR. Nucleic Acids Res 33(20): e179). All DNAoligonucleotides were synthesized by IDT.

Preparation of RNA and cDNA

Total RNA was isolated from spleens of 4-week-old Balb/C mice usingTrizol (Invitrogen) following the manufacturer's protocol. Total RNAs (1μg) was incubated with poly(A)polymerase (USB) molecules, therebygenerating polyadenylated microRNAs at the ′3 end. The polyadenylatedRNAs (20 μl) reverse transcribed using 2 μM RTUloop primer, 0.25 mM eachof dNTPs, 100 units Smartscribe reverse transcriptase, lx reversetranscriptase buffer, and 10 mM DTT (Clontech Laboratories). Thereactions were incubated at 42° C. for 90 minutes, and then at 95° C.for 5 minutes, to inactivate reverse transcriptase molecules and todegrade RNAs. All reverse transcriptase reactions included no-templateand minus-RT as controls.

Real-Time PCR

1 μl 10-time serially diluted reverse transcription reactions were usedfor real-time PCR, using the target miRNA as the forward primer and QRTUas the universal reverse primer. All the reactions were run on the ABI7900HT System (Applied Biosystems). Real-time PCRs were carried out in a20 μl reaction in triplicate.

Amplification curves were generated with an initial denaturing step at95° C. for 30 seconds, followed by 40 cycles at 95° C. for 5 seconds,and at 60° C. for 30 seconds. Dissociation curves were generated usingthe following programs: PCR products were denatured at 95° C. for 15seconds, cooled to 60° C. for 15 seconds, and finally at 95° C. for 15seconds.

All QPCRs were carried out using Premix Ex Taq™ or SYBR Premix II Ex Taq(Perfect Real Time) (Clontech Laboratories). SYBR Green I and TaqMan®PCR products were visualized on 2% agarose gels by Grow-Green staining(eEnzyme).

Example 2 Reproducibility of the UPR MicroRNA qRT-PCR Assay

The amplification efficiency is critical a reproducible qRT-PCR assay.To examine the reproducibility of the UPR miRNA qRT-PCR assay,amplification efficiency was calculated using the relative standardcurve method (FIGS. 2B&C). Briefly, qRT-PCR cDNA templates of miRNAswere prepared by 10-time serial dilution, which is equivalent to 10, 1,0.1, 0.01 and 0.001 ng of total RNA, respectively. Each sample was runin triplicate.

The standard curves were generated using the Prism HT7900 system. Thecorrelation coefficient (R2) values were greater than 0.99 (except forthat of miR-150, which is 0.98), indicating excellent linear reliabilitybetween RNA concentration and the C_(T) value of reverse transcriptionreal-time PCR reaction for each miRNA.

PCR efficiency was calculated by the formula E=−1+10^(−1/slop). FIGS.2A&B show the amplification curve plot and standard curve plot of themiR-690 qRT-PCR. The PCR efficiency is 0.94 for miR-690 and 1.1 formiR-142 and miR-150 (efficiencies between 0.90 and 1.10 are typicallyacceptable).

Amplification curves correlated to the concentration of RNA template andspanned five orders of magnitude (FIGS. 2A&B). The CT values from theamplification curves for the 96 miRNA QPCR array range from 17 to 35,equivalent to five orders of magnitude (FIG. 2D).

Example 3 Specificity of the UPR MicroRNA qRT-PCR Assay

Without the use of DNases, no RNA isolation method can consistentlyproduce RNA free from genomic DNA contamination. This Example testswhether residual genomic DNA contamination may cause non-specificity ofthe UPR miRNA qRT-PCR assay.

Briefly, mouse tail genomic DNA, isolated by the DNeasy Blood & TissueKit (QiaGen), was used as the template in the UPR miRNA qRT-PCR assay.Mouse tail genomic DNA was added during the polyadenylation reaction(0.3 μg genomic DNA used), or QPCR reactions as templates (1 μg genomicDNA used).

The results show no detectable signals in the UPR miRNA QPCR assay, whenusing genomic DNA either as a sham control for the total RNA (FIG. 3B),or as the direct template of QPCR (FIG. 3C). The agarose gelelectrophoresis shows nonspecific amplification (FIG. 3D), which may bedetected by SYBR Green. The amount of the genomic DNA used in thisExample far exceeds residual DNA that may be present in total RNApreparation. This indicates that the accuracy of the UPR miRNA qRT-PCRassay will not be affected by genomic DNA contamination in total RNApreparation.

Total RNA was also used as the template of UPR miRNA qRT-PCR assaywithout the polyadenylation step. This investigates whether mRNAs, themajor component of total RNA, will produce significant non-specificsignals in the assay. When polyadenylated mRNA is used as the templateof the qRT-PCR assay, the CTs (threshold cycle) of qRT-PCR assays aregreater than 38 (FIG. 3A). CT values greater than 35 approaches thesensitivity limits of the real-time PCR detection system of miRNAs. Thissuggests that the contribution of the background signals from mRNA inthis assay is negligible.

Example 4 Detection of Non-Specific Amplification by MicroRNA SYBR GreenqPCR Array

SYBR Green is a fluorescent dye. It non-specifically intercalates intodouble-stranded DNA and detects double-stranded DNA.

FIG. 4 shows detection of non-specific amplification by 96-miRNA qRT-PCRarray using SYBR Green. The dissociation curves (FIG. 4A) show that mostmiRNA qRT-PCRs produced a small peak and a major peak. The small peakrepresents non-specific amplification, while the major peak representsamplification of the desired PCR products. The log amplification curveplot (FIG. 4B) shows that there are non-exponent amplifications for lowabundant miRNAs. The results suggest that the SYBR Green assay mayover-detect miRNAs, which is present in low amounts.

Example 5 Use of Multiple Hydrolysis Locked-Nucleic Acid (LNA) ProbesImproves Sensitivity of UQmiR RT-qPCR Assay

In this Example, multiple hydrolysis locked-nucleic Acid (LNA) probesare used to sensitively detect amplicons of the amplification reaction(FIG. 5). Use of a plurality of probes significantly enhancessensitivity for miRNA profiling and the increase in sensitivity equalsto the number of probes used minus 1. This technique is especiallyuseful for detecting low copy of miRNAs and/or detecting miRNAs in smallsamples. For example, miRNAs present in samples obtained from lasercapture microdissection microscopy (LCMM) or a single cell can bedetected using the present probe design. Such low copy of miRNAs cannotbe detected using the commercially-available miRNA detection methods.

FIG. 7 shows that the UQmiR assay of the present invention is moresensitive than the Taqman RT-qPCR assay, which is the most sensitive andspecific miRNA assay currently available.

Example 6 Use of UQmiR Forward Primer Enhances Specificity of UQmiRRT-qPCR Assay

This Example illustrates one embodiment of the forward primer(UQmiRprimer) useful for the amplification reaction of the present miRNAassay. In one embodiment, the forward primer comprises the target maturemiRNA sequence and additional nucleotides attached to the 5′ and 3′ endsof the mature miRNA sequences.

As shown in FIG. 6, by attaching additional sequences at the 5′ and3′-ends of the mature miRNA sequences, the UQmiR primers specificallybind to the cDNAs of mature miRNAs and extension occurs at both ends.The forward primer does not anneal to non mature miRNA sequences such asgenomic DNA, cDNAs of pre-miRNA, pri-miRNA and mRNA that comprise themature miRNA sequence; therefore, no extension occurs in non maturemiRNA sequences.

UQmiR primers that comprise extra sequences at the 3′-end of the miRNAsequence discriminate mature miRNAs over pre-miRNAs in 50 folds (FIG.8A-B). UQmiR primers that comprise extra sequences at both 3′- and5′-ends of the mature miRNA sequence discriminate mature miRNAs overpre-miRNAs over one thousand times; thus, the present miRNA assay is farmore specific than the ABI miRNA Taqman assay (FIG. 9A-C).

Example 7 Specificity and Sensitivity of the UQmiR RT-qPCR Assay

FIG. 10 shows that the RT-qPCR miRNA Taqman assay (UQmiR) assaydiscriminates miRNAs within the mouse Let-7 miRNA family. The miRNAmembers of the same family are highly homologous and can differ fromeach other only in terms of a few bases. Also, the UQmiR miRNA qPCRassay is as sensitive as the commercially available Taqman assay (FIG.12).

In addition, the UQmiR can sensitively detect plasma and serum miRNAs inclinical samples. FIG. 12 shows that the present miRNA assay sensitivelyand specifically detect 88 out of 94 miRNAs in 0.8 μl of plasma samples.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims. In addition, anyelements or limitations of any invention or embodiment thereof disclosedherein can be combined with any and/or all other elements or limitations(individually or in any combination) or any other invention orembodiment thereof disclosed herein, and all such combinations arecontemplated with the scope of the invention without limitation thereto.

All patents, patent applications, provisional applications, andpublications referred to or cited herein, supra or infra, areincorporated by reference in their entirety, including all figures andtables, to the extent they are not inconsistent with the explicitteachings of this specification.

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We claim:
 1. A method for detecting, quantifying, and/or profiling atarget miRNA, comprising: a) contacting a sample containing miRNAs withan effective amount of poly(A)polymerase molecules to yield 3′end-polyadenylated miRNA molecules, b) contacting the sample with aneffective amount of a universal primer for reverse transcription andreverse transcriptases, and reverse transcribing the polyadenylatedmiRNA molecules to yield corresponding c-DNA molecules; and c)contacting the sample with an effective amount of a universal reverseprimer and a target miRNA-specific forward primer, and amplifying thecorresponding c-DNA molecules using an amplification reaction; whereinthe universal primer for reverse transcription is an oligonucleotidecomprising: a (dT)_(n) sequence flanked by a stem-looped universaladaptor sequence, wherein “n” is an integer ranging from 8 to 50,wherein the universal primer comprises at least two nucleotides adjacentto the 3′ end of the (dT)_(n) sequence, and the nucleotide immediatelyadjacent to the (dT)_(n) sequence is not T, wherein the universaladaptor sequence near the 5′ end of the (dT)_(n) sequence forms into astem-loop structure by base-pairing, and wherein the universal reverseprimer in the amplification reaction is an oligonucleotide comprising asequence that is, or base-pairs with, at least part of the adaptorsequence near or toward the 5′ end of the (dT)_(n) sequence.
 2. Themethod, according to claim 1, wherein the forward primer comprises thetarget miRNA sequence and three or more additional nucleotides attachedto the 3′ end of the target mature miRNA sequence.
 3. The method,according to claim 1, wherein the additional nucleotides attached to the3′ end of the target mature miRNA sequence are adenine molecules.
 4. Themethod, according to claim 2, wherein one or more additional nucleotidesare attached to the 5′ end of the target mature miRNA sequence.
 5. Themethod, according to claim 1, wherein the amplification reaction forcDNA amplification is quantitative real time polymerase chain reaction(qRT-PCR).
 6. The method, according to claim 1, comprising: contactingthe sample with a sufficient amount of a universal probe, detectingand/or quantifying a level of amplified c-DNA molecules, and determiningthe level of the target miRNA based on the c-DNA level; wherein theuniversal probe comprises a sequence that is, or base-pairs with, atleast part of the adaptor sequence near the 5′ end of the (dT)_(n)sequence.
 7. The method, according to claim 6, wherein the universalprobe comprises locked nucleic acid (LNA) molecules.
 8. The method,according to claim 6, wherein the universal probe comprises a detectablemoiety.
 9. The method, according to claim 8, wherein the detectablemoiety is a fluorophore selected from FAM, CY5, CY3, BODIPY FL, TEXASRED, or any combination of two or more of the foregoing.
 10. The method,according to claim 6, wherein at least two universal probes are used.11. The method, according to claim 1, wherein the amplification reactionfor amplification of cDNA molecules is performed once.
 12. The method,according to claim 1, wherein the threshold cycle (Ct) of theamplification reaction for amplification cDNA molecules ranges from 17to
 35. 13. The method, according to claim 1, wherein the sample is atotal RNA sample.
 14. The method, according to claim 1, wherein themethod is capable of detecting, quantifying and/or profiling microRNA ina total RNA sample of about 1 pg.
 15. The method, according to claim 1,wherein the universal reverse primer comprises dUTP.
 16. The method,according to claim 15, further comprising adding uracil-DNA Glycosylasemolecules to the amplification reaction mixture.
 17. The method,according to claim 1, comprising adding nuclease molecules to thereaction mixture.